Analysis Of Drug Resistance And Detection Of Drug Resistance Gene And Class ? Integron In Porcine ExPEC

Extraintestinal pathogenic Escherichia coli(Ex PEC)can not only colonize in the intestine normally,but also invade blood vessels,cause bacteremia,and induce sepsis or local extraintestinal infection,such as meningitis,arthritis,pneumonia,endocarditis,etc.In recent years,the detection rate of pig Ex PEC has been increasing year by year in China.Due to the increase of incidence rate,mortality and weight loss,and the increase of treatment,vaccine and feed additives,the economic losses caused by Ex PEC caused by colibacillosis of pigs have become increasingly significant.At present,antibiotics are still mainly used in the prevention and treatment of Ex PEC disease,but with the extensive use of antibiotics,the number of multidrug-resistant Ex PEC is increasing,and even many strains have evolved into superbacteria,which seriously affects the selection of effective antibiotics for the prevention and treatment of diseases caused by Ex PEC,and brings great hidden danger to the safety of human and animals.In this study,we conducted an epidemiological survey of swine bacterial disease in family farms of Anhui,Jiangsu,Shandong,Hebei,Guangxi and other five provinces of a group company in the early stage of the laboratory Detection and analysis of drug resistance genes and class I integron:(1)the susceptibility to 22 kinds of antibiotics was determined by disk agar diffusion method(K-B method),and the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of 22 kinds of antibiotics were determined by micro broth dilution method.(2)According to the results of drug sensitivity test,the related drug resistance genes were identified,and the chromosomal DNA and plasmid DNA were detected by PCR.(3)PCR was used to detect class I integron in chromosome DNA and plasmid DNA.The purpose of this study was to understand the drug sensitivity and drug resistance characteristics of porcine Ex PEC,so as to provide technical support for effectively controlling the threat of Ex PEC to pigs,reducing the generation and spread of drug resistance of Ex PEC,and ensuring the continuous,stable and healthy production of pigs.The results showed that:(1)disk agar diffusion method(K-B method): 54 strains of porcine Ex PEC had the highest resistance rate to penicillin(100%);the highest sensitivity rate to polymyxin B(75.93%);the resistance rates to trimethoprim,compound sulfamethoxazole,amoxicillin,ampicillin,doxycycline,tetracycline and Cefalexin were more than 80%;the resistance rates to florfenicol,streptomycin and kanamycin were more than 80% The drug resistance rates of cefazolin,cefazolin,spectinomycin,gentamicin,ceftriaxone,neomycin,enrofloxacin,ofloxacin,amikacin,polymyxin E and ciprofloxacin ranged from 27.48% to 79.63%.The 54 strains of swine derived Ex PEC were resistant to more than 6 kinds of antibiotics,which were multi drug resistance.Among them,15 strains were resistant,accounting for 18.52%(10 / 54strains),and 44 strains were resistant.Micro broth dilution method: 54 strains of porcine Ex PEC were most resistant to penicillin,trimethoprim,compound sulfamethoxazole,florfenicol,ampicillin and amoxicillin(MIC > 256 ?g/m L,no MBC value),sensitive to polymyxin B(MIC between 2-8 ?g/m L,MBC values were 4,8,16,32 and 64 ?g/m L),and resistant to cefazolin,tetracycline,Cefalexin,spectinomycin and streptomycin The tolerance rates of the five antibiotics were more than 80%(MIC values were between? 4-> 256?g/m L,MBC values were 8,16,32,62,128 and 256 ?g/m L,respectively,and no MBC value was found for strains with MIC values > 256?g/m L).The 54 strains of swine derived Ex PEC could tolerate more than 11 kinds of antibiotics,which were multi drug resistance.Among them,17 kinds were mainly tolerated,accounting for18.52%(10/54 strains),and there were 47 kinds of drug resistance spectrum.(2)According to the results of drug sensitivity test,20 genes were identified.In chromosome DNA,except for aph(3 ')-IIa gene,19 kinds of drug-resistant genes could be detected,among which aad A1(87.04%)and tet A(87.04%)had the highest detection rate,and qnr A(3.7%)had the lowest detection rate;all strains carried more than 5 kinds of drug-resistant genes,up to 13 kinds,among which 10 kinds accounted for the highest proportion,up to 24.08%(13/54 strains),and sul1 genes mainly existed in them.In plasmid DNA,except tet B,tet C,SHV and qnr A genes,16 kinds of resistance genes could be detected.The detection rates of flo R(98.15%)and Par C(98.15%)were the highest,and the detection rates of aph(3 ')-IIa(3.7%)and OXA(3.7%)were the lowest.All strains carried more than 5 kinds of resistance genes,up to 13 kinds,of which 10 kinds accounted for the highest proportion,up to 27.78%(15/54 strains),while flo R,tet M,aph(3')-IIa,aad B and mcr-1 genes mainly exist in them.(3)Among 54 strains of swine derived Ex PEC,52 strains of class I integron were detected by chromosome DNA,with a positive detection rate of 96.3%,and 53 strains of class I integron were detected by plasmid DNA,with a positive detection rate of 98.15%.Class I integron positive strains were multi drug resistant.The results showed that:(1)The disk agar diffusion method(inhibition zone diameter determination)and micro broth dilution method(MIC and MBC determination)were used to identify 54 strains of porcine Ex PEC with multiple drug resistance,and the drug resistance spectrum was complex and diverse.The latter method was more sensitive and accurate than the former method.(2)Among the 20drug-resistant genes detected,the chromosome DNA carrying rate of 54 strains of porcine Ex PEC was 95%,aminoglycosides and tetracycline resistance genes aad A1 and tet A,and tet B,tet C,SHV and qnr A genes mainly existed in them;plasmid DNA carrying rate was 80%,mainly flo R and Par C,and aph(3 ')-IIa genes mainly existed in them.There was a certain correlation between the occurrence of drug resistance and the existence of drug resistance genes,and all strains carried multiple drug resistance genes.(3)The positive rates of class I integrons in genomic DNA and plasmid DNA were 96.3%and 98.15% respectively.The existence of class I integrons was closely related to the multidrug resistance of strains,and the existence of class I integrons increased the risk of horizontal transfer of drug resistance genes.